The Na-CI cotransporter (NCC) is expressed in the apical membrane of the distal convoluted tubule and is responsible for the reabsorption of 5% -10% of filtered Na and Cl. NCC is the target of the thiazide diuretics prescribed very frequently for treatment of hypertension, edema and heart failure. NCC abundance is highly regulated by: dietary NaCI restriction, aldosterone, estrogen and thiazide treatment, additionally, NCC is mutated in Gitelman's syndrome leading to salt wasting, hypokalemia and metabolic alkalosis, all confirming a homeostatic role of NCC abundance in regulating Na balance and blood pressure. We recently tested the hypothesis (in rats in vivo) that high salt diet provokes redistribution of DCT NCC from apical plasma membrane (PM) to sub-apical intracellular membrane pools (IM) and that salt restriction favors redistribution to the PM. Applying a density gradient centrifugation approach discovered that NCC redistributes to higher density membranes in response to high salt diet and to lower density membranes during salt restriction. By EM, sub-apical IM pools of NCC were evident and the ratio of NCC labeling in the IM to PM was higher in high salt diet than low salt diet where most all the NCC is in the PM, supporting our hypothesis. Our provocative preliminary results set the groundwork for addressing the molecular mechanisms regulating NCC subcellular distribution in vivo. In this R21 application we aim to fully confirm the hypothesis that NCC subcellular distribution is chronically regulated by salt diet and proceed to test the hypothesis that NCC distribution in PM vs. IM can be acutely regulated by adrenergic stimulation and/or acute hypertension. Our studies will break new ground in determining: the role of NCC in responding to adrenergic (antinatriuretic) and blood pressure (natriuretic) signals, the source and destination of NCC, the NCC associated proteins en route. In order to define NCC protein-protein interactions and how they change with apparent NCC redistribution in each model we need to generate reagents and methods to immunoprecipitate NCC along with associated proteins and along with NCC containing vesicles for subsequent candidate and proteomic analyses. This line of investigation aims to provide definitive evidence for regulated trafficking of NCC in vivo, identify potential mediators of the NCC trafficking, identify NCC trafficking defects in disease, and suggest alternative mechanisms to alter NCC therapeutically. [unreadable] [unreadable] [unreadable]